CN105942286A - Sea cucumber functional health-care products and preparation method thereof - Google Patents
Sea cucumber functional health-care products and preparation method thereof Download PDFInfo
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- CN105942286A CN105942286A CN201610273191.8A CN201610273191A CN105942286A CN 105942286 A CN105942286 A CN 105942286A CN 201610273191 A CN201610273191 A CN 201610273191A CN 105942286 A CN105942286 A CN 105942286A
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- stichopus japonicus
- taurine
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/616—Echinodermata, e.g. starfish, sea cucumbers or sea urchins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
- A61K31/355—Tocopherols, e.g. vitamin E
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention discloses sea cucumber functional health-care products and a preparation method thereof. The sea cucumber functional health-care products are prepared from freeze-dried sea cucumber powder, taurine and vitamin E as main raw materials according to the recipe combining modern medicines and traditional Chinese medicinal theories. The freeze-dried sea cucumber powder can supplement kidney channels and benefit essence, is rich in proteins and amino acids which can promote the synthesis of immune substances, and can improve organism immune functions. The taurine and vitamin E also have the effects of improving immunity. The selected raw materials are wide in resources, reliable in quality, and affordable in prices. Besides, a large number of embodiment experimental studies show that the health-care products have good enhancing effects for body immune functions. Compared with other similar health-care food products, the health-care food products have certain comprehensive advantages.
Description
Technical field
The invention belongs to technical field of functional food, be specifically related to a kind of Stichopus japonicus functional health care product that can improve body immunity and
Its preparation method.
Background technology
The body constitution of people is worse and worse now, and the state of subhealth state gets more and more.Improving resistance is that the modern times, everyone was concerned about
Topic, the principal character of subhealth state includes: all symptoms that 1. on body and mind, unconformable sensation is reflected, as tired,
Weakness, mood change etc., its situation was difficult to clearly within suitable period；2. incompatible with age organizational structure or physiology merit
The caused various weaknesses that can go down show；3. Dysbiosis state；4. the sick front physiology pathological change of some disease.
Clinical manifestation is varied, and body aspect can behave as fatigue and weak, muscle and joint aches, dizzy headache, cardiopalmus breast
Vexed, sleep disordered, inappetence, gastral cavity abdomen discomfort, loose stool constipation, sexual hypofunction, it is afraid of cold that To Be Protected from Heat, is prone to flu, eye
Dry and astringent etc.；Psychological aspects can be presented with depressed, upset, lather, being irritable and getting angry easily, frightened timid, memory
Decline, attention can not be concentrated, deficient in energy, bradykinesia etc.；Social communication's aspect can be presented with can not preferably undertaking phase
The social role answered, work, learning difficulty, it is impossible to normally handle inter personal contact, family relation well, it is difficult to carry out normal
Social communication etc..
World Health Organization (WHO) is early it has proven convenient that repeated cold is a kind of performance of hypoimmunity.In other words, if a people exists
In 1 year, flu is more than 5 times, and the ability resisting various major disease just reduces by 80% than normal person, and this kind of crowd also can be along with
Some other chronic disease.The people of China more than 70% is all in sub-health state, due to the increase of operating pressure, nutrition
The factor such as increase the weight of unbalance, that pollute, causes immunity of organisms generally to reduce, thus become various sub-health state latent because of.Exempt from
Epidemic disease power is the basis of human body resist the disease and treatment disease, and hypoimmunity can cause multiple disease, as caught a cold, and hepatitis, even
Tumor etc..
According to the investigation and analysis of China's treatment status of blood lipids disorders, the age of China's hyperlipidemia patient mainly between 40～80 years old,
Wherein 60～account for 40.9% in 69 years old, in accompanying diseases, hypertension is most common, accounts for 60.6%；Secondly coronary heart disease, account for 45.1%,
Diabetes 32.8%, apoplexy 13.7%.Hyperlipemia prevalence mainly shows as developed area higher than economics of underdevelopment ground
District, the prevalence of some crowd's hypercholesterolemias and high low density lipoprotein is more than 30%, and have is even more than 50%.
The main cause causing above-mentioned sub-health status has a lot, including: diet is unreasonable, shortage is moved, it is irregular to work and rest,
Do not have enough sleep, big, the long-term unhealthy emotion of psychentonia, mental pressure etc..Therefore, in modern society, the most artificially
Allow and oneself choose the health product improving resistance faster away from sub-health state.Health product have become as modern and improve opposing
One critically important means of power；
Under normal circumstances, health product in the market, it is broadly divided into several big class: with vitamin and mineral as main component
Auxotype health product；With natural or rare species as raw material, extract the health product of effective nutritional labeling；With rare Chinese medicine or have
The animals and plants of medical value are the tonic type health product of primary raw material；The health product that effective component extracting is made from marine organisms；
The health product made for raw material with animal foremilk；Health product based on " the 7th nutrient " dietary fiber；Pure natural, no
Aminoacid containing any additive is main health product.And all kinds of health product are because raw material is different with processing mode, therefore have
Its respective feature and the limitation of application, for the market demand of abundant diversification day by day, abundant health product kind, the present invention
A kind of new health care product is provided.
Summary of the invention
The present invention is with lyophilized Stichopus japonicus powder, taurine, vitamin E as primary raw material, according to modern medicine and Traditional Chinese Medicine theory
Combine and prescription.Concrete, the preparation method of the herein described Stichopus japonicus functional health care product that can improve body immunity is as follows:
1, lyophilized Stichopus japonicus powder: Stichopus japonicus is carried out, after roguing, by Stichopus japonicus after multistage enzymolysis, centrifugal, hyperfiltration treatment straight
Connect vacuum lyophilization.
Concrete grammar: the Stichopus japonicus by being carried out, after roguing grinds to form screened stock shape, according to Stichopus japonicus and water 1:(0.5-3) quality
Than mixing, after crossing 100-200 eye mesh screen, holothurians autoenzyme, neutral protease, alkaline protease is utilized to carry out three grades of falls successively
Solve.By enzymolysis solution by centrifugal segregation insoluble matter, then carry out hyperfiltration treatment, obtain Stichopus japonicus aqueous solution, after being concentrated in vacuo,
Finally carry out vacuum lyophilization and obtain lyophilized Stichopus japonicus powder.
2, according to lyophilized Stichopus japonicus powder: taurine: the mass ratio of vitamin E is=(180-220): (30-40): the ratio of (10-30) is entered
Row is sufficiently mixed stirring；
3, by the lyophilized Stichopus japonicus powder of step 2 gained, taurine powder, tocopherol admixture after 40-100 mesh sieves, put mixed
Mixing 30min in conjunction machine, obtain mixed powder, mixed powder answers uniform color consistent, carries out filling capsule, being packaged as finished product.
Concrete, in technique scheme, step 1) described in by Stichopus japonicus through multistage enzymolysis method particularly includes: by Stichopus japonicus
Grind to form screened stock shape, according to Stichopus japonicus and water 1:(0.5-3) ratio mix, cross after 100-200 eye mesh screen, utilize successively
Holothurians autoenzyme, neutral protease, alkaline protease carry out multi-stage degradation.
Concrete, in technique scheme, described holothurians autoenzyme, neutral protease, the use condition of alkaline protease be:
Holothurians autoenzyme, neutral protease, alkaline protease carry out multi-stage degradation at 40 DEG C, 45 DEG C, 55 DEG C respectively, and described Stichopus japonicus is certainly
Lyase, neutral protease, the usage amount of alkaline protease are respectively as follows: the mass ratio of 0.1-2%, 0.1-0.6%, 0.02-0.07%.
Concrete, in technique scheme, step 1) described in centrifugal condition be: enzymolysis solution is passed through 3000-10000rpm
Centrifugal 10-40min.
Concrete, in technique scheme, step 1) described in Stichopus japonicus be in fresh and alive sea cucumbers, dry Stichopus japonicus, salted sea cucumber
One, and protein content is more than 50%.
Another aspect of the present invention is, protects a kind of Stichopus japonicus functional health care product utilizing method as discussed above to prepare, color
Pool uniformity, and be grey-brown powder.And a kind of utilize health product mentioned above improve in terms of body immunity should
With.In the method for the invention, Stichopus japonicus is the water-soluble substances through digestion process, and protein and various active composition hold very much
Easily digested, and in enzymolysis process, form some small peptide isoreactivity materials.The Stichopus japonicus of bad absorption is changed poling
The Stichopus japonicus aqueous solution of absorption easy to digest, then it is equipped with taurine and vitamin E, demonstrate,proved by the function test of the embodiment of the present invention 4
Bright, the nutrient substance of Stichopus japonicus has obtained effective utilization, and taurine, vitamin E and sea cucumber nutrient composition with the use of reaching
Having arrived more preferable synergy, this product has extremely strong health care.
Detailed description of the invention
Following nonlimiting examples can make those of ordinary skill in the art that the present invention is more fully understood, but not with any side
Formula limits the present invention.
Raw material used by the embodiment of the present invention 1～3 the most all can be prepared by conventional methods or commercial sources is bought
Obtain, wherein:
Taurine: the prescription of GB14759 national food safety standard food additive taurine should be met.
Vitamin E: GB29942 national food safety standard food additive vitamin E (dl-alpha-tocopherol) should be met
Matter；
Holothurians autoenzyme, neutral protease, alkaline protease can be bought by commercial sources respectively and obtain.
Described Stichopus japonicus is the one in fresh and alive sea cucumbers, dry Stichopus japonicus, salted sea cucumber, and protein content is more than 50%.
Embodiment 1
1, lyophilized Stichopus japonicus powder: be carried out by fresh and alive sea cucumbers, after roguing, by Stichopus japonicus through multistage enzymolysis, centrifugal, hyperfiltration treatment
Rear directly vacuum lyophilization.
Concrete grammar: Stichopus japonicus is ground breaking into screened stock shape, according to Stichopus japonicus by the Stichopus japonicus colloid mill by being carried out, after roguing
Mix with the ratio of water 1:1, after crossing 100 eye mesh screens, utilize holothurians autoenzyme, neutral protease, basic protein successively
Enzyme carries out multi-stage degradation at 40 DEG C, 45 DEG C, 55 DEG C respectively.By enzymolysis solution by, after centrifugal segregation insoluble matter, carrying out hyperfiltration treatment,
Obtain Stichopus japonicus aqueous solution, after being concentrated in vacuo, finally carry out vacuum lyophilization and obtain lyophilized Stichopus japonicus powder.
Described holothurians autoenzyme, neutral protease, the usage amount of alkaline protease are respectively as follows: the matter of 0.1%, 0.1%, 0.02%
Amount ratio.By the centrifugal condition of enzymolysis solution it is: 3000rpm is centrifuged 40min；
2, according to lyophilized Stichopus japonicus powder: taurine: the mass ratio of vitamin E is that the ratio of 180:30:10 carries out being sufficiently mixed stirring；
3, by the lyophilized Stichopus japonicus powder of step 2 gained, taurine powder, tocopherol admixture after 40-100 mesh sieves, put mixed
Mixing 30min in conjunction machine, obtain mixed powder, mixed powder answers uniform color consistent, carries out filling capsule, being packaged as finished product.Sample is checked and approved
Net content is 0.25g/ grain.Capsule 's content is grey-brown powder.Human body recommended dose is: 1.5g/60kgBW, puts cool place, does
Dry place preserves, storage life 24 months.
Embodiment 2
1, lyophilized Stichopus japonicus powder: be carried out by dry Stichopus japonicus, after roguing, by Stichopus japonicus after multistage enzymolysis, centrifugal, hyperfiltration treatment
Directly vacuum lyophilization.
Concrete grammar: Stichopus japonicus is ground breaking into screened stock shape, according to Stichopus japonicus by the Stichopus japonicus colloid mill by being carried out, after roguing
Mix with the ratio of water 1:2, after crossing 150 eye mesh screens, utilize holothurians autoenzyme, neutral protease, basic protein successively
Enzyme carries out multi-stage degradation at 40 DEG C, 45 DEG C, 55 DEG C respectively.By enzymolysis solution by, after centrifugal segregation insoluble matter, carrying out hyperfiltration treatment,
Obtain Stichopus japonicus aqueous solution, after being concentrated in vacuo, finally carry out vacuum lyophilization and obtain lyophilized Stichopus japonicus powder.
Described holothurians autoenzyme, neutral protease, the usage amount of alkaline protease are respectively as follows: the quality of 1%, 0.3%, 0.05%
Ratio.By the centrifugal condition of enzymolysis solution it is: 5000rpm is centrifuged 20min；
2. according to lyophilized Stichopus japonicus powder: taurine: the mass ratio of vitamin E is that the ratio of 200:35:15 carries out being sufficiently mixed stirring.
3, by the lyophilized Stichopus japonicus powder of step 2 gained, taurine powder, tocopherol admixture after 40-100 mesh sieves, put mixed
Mixing 30min in conjunction machine, obtain mixed powder, mixed powder answers uniform color consistent, carries out filling capsule, being packaged as finished product.Sample is checked and approved
Net content is 0.25g/ grain.Capsule 's content is grey-brown powder.Human body recommended dose is: 1.5g/60kgBW.Put cool place, do
Dry place preserves, storage life 24 months.
Embodiment 3
1, lyophilized Stichopus japonicus powder: be carried out by salted sea cucumber, after roguing, by Stichopus japonicus through multistage enzymolysis, centrifugal, hyperfiltration treatment
Rear directly vacuum lyophilization.
Concrete grammar: Stichopus japonicus is ground breaking into screened stock shape, according to Stichopus japonicus by the Stichopus japonicus colloid mill by being carried out, after roguing
Mix with the ratio of water 1:3, after crossing 200 eye mesh screens, utilize holothurians autoenzyme, neutral protease, basic protein successively
Enzyme carries out multi-stage degradation at 40 DEG C, 45 DEG C, 55 DEG C respectively.By enzymolysis solution by, after centrifugal segregation insoluble matter, carrying out hyperfiltration treatment,
Obtain Stichopus japonicus aqueous solution, after being concentrated in vacuo, finally carry out vacuum lyophilization and obtain lyophilized Stichopus japonicus powder.
Described holothurians autoenzyme, neutral protease, the usage amount of alkaline protease are respectively as follows: the quality of 2%, 0.6%, 0.07%
Ratio.By the centrifugal condition of enzymolysis solution it is: 1000rpm is centrifuged 10min；
2, according to lyophilized Stichopus japonicus powder: taurine: the mass ratio of vitamin E is that the ratio of 220:40:20 carries out being sufficiently mixed stirring.
3, by the lyophilized Stichopus japonicus powder of step 2 gained, taurine powder, tocopherol admixture after 40-100 mesh sieves, put mixed
Mixing 30min in conjunction machine, obtain mixed powder, mixed powder answers uniform color consistent, carries out filling capsule, being packaged as finished product.Sample is checked and approved
Net content is 0.25g/ grain.Capsule 's content is grey-brown powder.Human body recommended dose is: 1.5g/60kgBW, puts cool place, does
Dry place preserves, storage life 24 months.In following Example 4, sample ID summary is Stichopus japonicus taurine vitamin E capsule.
Embodiment 4
One. experiment material
Select Beijing HFK Bio-Technology Co., Ltd. [credit number: SCXK-(capital) 2009-0007] breed 18～22g
Kunming kind health cleaning grade female mice 192, is divided into four batches and tests, and every batch is randomly divided into 4 groups, often group 12.
Test and a collection of carry out internal organs/weight ratio pH-value determination pH, delayed allergy experiment, half hemolysis value (HC50) mensuration and antibody
The mensuration of cellulation number；Test two batches and carry out carbonic clearance experiment；Test three batches carry out Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red carefully
Born of the same parents test；Test four batches of mouse lymphocyte transformation experiments carrying out ConA induction and NK cytoactive detection.Laboratory animal
Raising in SPF level animal housing of Health Food Function Detection Center, Applied Literature and Science College, B, laboratory animal uses license
Card number: SYXK (capital) 2007-0020.Normal feedstuff is by Beijing HFK Bio-Technology Co., Ltd. [credit number: SCXK-
(capital) 2009-0008] produce.
Dosage: recommended dose for be equivalent to for adult (by 60kg weighing machine) 1.5g every day, be equivalent to 0.025g/ day/kgBW.
Experiment relates to the 5 of human body recommended amounts, 10,30 times respectively, i.e. every day: 0.12g/kgBW, 0.25g/kgBW and 0.75g/kgBW
For basic, normal, high dosage group.Tested material sterilized water is prepared.Per os gives tested material once a day, and continuous gavage was surveyed after 33 days
Indices.Mouse stomach volume is 0.10mL/10g Mus weight.Set a solvent control thing (0g/kgBW) simultaneously, use sterilized water
Replacing tested material, every day, gavage volume was identical with each tested material group.Each dosage group all gives normal feedstuff.
The data processing method of the embodiment of the present invention 4 is:
Data process is carried out with SPSS software.Use variance analysis, but need to first carry out homogeneity of variance inspection by the program of variance analysis
Testing, variance is neat, calculates F value, F value < F0.05, conclusion: no significant difference between each group mean；F value >=F0.05, P≤0.05,
Add up with the comparative approach two-by-two of mean between multiple experimental grouies and a matched group；The data of abnormal or heterogeneity of variance are entered
Row suitable variable conversion, after meeting normal state or variance requires together, adds up by the data after conversion；If after variable conversion
Still it is not up to normal state or the neat purpose of variance, uses rank test instead and add up.
Two. experimental technique
1. the mensuration of organ weight ratio value
After mouse weights, cervical dislocation is put to death, and takes spleen and thymus, removes most fascia, blots organ surface blood stains with filter paper, weigh,
Calculate spleen weight ratio and thymus body weight ratio.
The impact on Mouse Weight of the Stichopus japonicus taurine vitamin E capsule
The original body mass of mice respectively organized by table 1
From table 1, the original body mass of mice compares with between 0g/kgBW group four batches of laboratory animal each dosage groups, and difference is all without aobvious
Work property (P > 0.05).The i.e. original body mass of mice more equalizes between each group.
The impact on Mouse Weight of the table 2 Stichopus japonicus taurine vitamin E capsule
From table 2, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, and the body weight of mice exists
Four batches of each dosage groups compare with between 0g/kgBW group, and there are no significant for difference (P > 0.05).I.e. Stichopus japonicus taurine vitamin E glue
Mouse Weight is had no adverse effects by capsule.
The impact on mice organs/body weight ratio of the Stichopus japonicus taurine vitamin E capsule
The impact on mouse spleen/body weight ratio of the table 3 Stichopus japonicus taurine vitamin E capsule
From table 3, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, each dosage group spleen
/ body weight ratio compares with 0g/kgBW group, and there are no significant for difference (P > 0.05).I.e. Stichopus japonicus taurine vitamin E capsule is to little
The spleen of Mus/body weight ratio is without impact especially.
The impact on mouse thymus/body weight ratio of the table 4 Stichopus japonicus taurine vitamin E capsule
From table 4, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, each dosage group thymus
/ body weight ratio compares with 0g/kgBW group, and there are no significant for difference (P > 0.05).I.e. Stichopus japonicus taurine vitamin E capsule is to little
The thymus of Mus/body weight ratio is without impact especially.
2 delayed allergies (DTH) experiment (the foot sole of the foot thickens method)
Taking Sanguis caprae seu ovis, brine 3 times, every Mus is through lumbar injection 2% (v/v, with normal saline) hematocrit
SRBC (2000r/min, 10min) 0.2mL, 4d after sensitization, measure left back sufficient sole of the foot portion thickness, and same position is measured three times, takes
Meansigma methods.Then at measuring point subcutaneous injection 20% (v/v, with normal saline) hematocrit SRBC20 μ L, in injection
Rear 24h measures foot sole of the foot portion thickness, represents the degree of DTH with the difference of foot sole of the foot thickness before and after attacking.The difference of given the test agent group
It is significantly higher than the difference of matched group, can determine that this experimental result positive.
The impact on mice delayed allergy (DTH) of the table 5 Stichopus japonicus taurine vitamin E capsule
*: compare with 0g/kgBW group and have significant difference
From table 5, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, 0.25g/kgBW
Comparing with 0g/kgBW group with 0.75g/kgBW group, swelling degree of the paw has significant difference (P < 0.05).I.e. Stichopus japonicus taurine dimension
Raw element E capsule all can improve the delayed allergy ability of mice in 0.25g/kgBW and 0.75g/kgBW group.
The mouse lymphocyte transformation experiment (mtt assay) of 3ConA induction
Aseptic take spleen, be placed in the little plate filling appropriate aseptic Hank ' s liquid, gently spleen ground with tweezers, make single carefully
Born of the same parents' suspension.Filter through 200 eye mesh screens, make cell suspension.Wash 3 times with Hank ' s liquid, be centrifuged 10min (1000r/min) every time.
Then by cell suspension in the complete culture solution of 1mL, microscopy counts, and adjusting cell concentration is 3 × 106/mL.Again by spleen
Cell suspension point holes adds in 24 well culture plates, does not has a hole 1mL, and a hole adds 75 μ LConA liquid and (is equivalent to wherein
7.5 μ g/ml), 5%CO2, as comparison, is put in another hole, cultivates 72h in 37 DEG C of CO2 incubators.Cultivation terminates front 4h, every hole
Suck supernatant 0.7mL gently, add the 0.7mL RPMI1640 culture fluid without calf serum, be simultaneously introduced MTT
(5mg/mL) 50 μ L/ hole.Continue to cultivate 4h.After cultivation terminates, every hole adds 1mL acid isopropyl alcohol, piping and druming mixing, makes
Purple crystal is completely dissolved.Then this liquid is moved in cuvette, colorimetric determination on 755 spectrophotometers, wavelength 570nm.
The multiplication capacity of lymphocyte deducts by the optical density value adding ConA hole and is not added with the optical density value in ConA hole and represents lymphocyte
Multiplication capacity.The optical density difference of given the test agent group is significantly higher than the optical density difference of matched group, can determine that this experimental result sun
Property.
The impact on mouse lymphocyte transformation experiment of the table 6 Stichopus japonicus taurine vitamin E capsule
*: compare with 0g/kgBW group and have significant difference
From table 6, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, 0.75g/kgBW
Group compares with 0g/kgBW group, and the multiplication capacity of lymphocyte has significant difference (P < 0.01).I.e. Stichopus japonicus taurine vitamin E
Capsule can improve the lymphopoiesis ability of mice in 0.75g/kg BW group.
The mensuration (Jerne improves slide method) of 4 antibody-producting cell numbers
Taking Sanguis caprae seu ovis, brine 3 times, every Mus is through lumbar injection 2% (v/v, with normal saline) hematocrit
SRBC0.2mL.Mice cervical dislocation after SRBC immunity 5 days is put to death, takes out spleen, grind spleen gently, make thin
Born of the same parents' suspension.Centrifugal (1000r/min) 10min, washes 2 times with Hank ' s liquid, finally by cell suspension in 8mLHank ' s liquid.
After agarose heating for dissolving, Hank ' the s liquid of concentration double with equivalent mixes, subpackage small test tube, often pipe 0.5mL, then to pipe
(v/v, with SA liquid prepare) hematocrit SRBC50 μ L, the splenocyte suspension 8 μ L that inside add 10%, rapidly after mixing, is poured into
On the slide of brush agarose thin layer, do parallel plate, after agar solidification, slide level is buckled and be placed on horse, put into carbon dioxide
37 DEG C of incubation 1h in incubator, then join in slide frame groove with the complement (1:8) of SA buffer dilution, continue incubation
1.5h, counts hemolysis plaque number.Represent with plaque number/full splenocyte.The plaque number of given the test agent group is significantly higher than matched group
Plaque number, can determine that this experimental result positive.
The impact on mouse antibodies cellulation number of the table 7 Stichopus japonicus taurine vitamin E capsule
*: compare with 0g/kgBW group and have significant difference
From table 7, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, 0.75g/kgBW
Group compares with 0g/kgBW group, and antibody-producting cell number has significant difference (P < 0.05).I.e. Stichopus japonicus taurine vitamin E glue
Capsule can improve the antibody-producting cell number of mice in 0.75g/kgBW group.
The mensuration of 5 half hemolysis value (HC50)
Taking Sanguis caprae seu ovis, brine 3 times, every Mus is through lumbar injection 2% (v/v, with normal saline) hematocrit SRBC0.2mL
Carry out immunity.After 5 days, extract eyeball and take blood in centrifuge tube, place about 1h, solidification blood is peeled off with tube wall, makes serum fill
Analyzing, 2000r/min is centrifuged 10min, collects serum.It is 300 times with SA buffer by serum-dilution, takes 1mL and put examination
In pipe, it is sequentially added into 10% (v/v uses SA buffer) hematocrit SRBC0.5mL, complement 1mL and (presses with SA buffer
1:8 dilutes).Separately set the control tube (replacing with SA buffer) of not increase serum.Put after 37 DEG C of waters bath with thermostatic control are incubated 15min,
Ice bath terminates reaction.2000r/min is centrifuged 10min, takes supernatant 1mL, adds Dou Shi reagent to 3mL.Take 10% (v/v. simultaneously
Use SA buffer) hematocrit SRBC0.25mL, add Dou Shi reagent to 4mL in another test tube, fully mix, place
After 10min, sentence control tube in 540nm and make blank, measure each pipe optical density value respectively.The amount of hemolysin is with half hemolysis value
(HC50) represent, be calculated as follows:
Optical density value × extension rate during sample half hemolysis value=sample optical density value/SRBC HD50
The HC50 of given the test agent group is significantly higher than the HC50 of matched group, can determine that this experimental result positive.
Table 8 Stichopus japonicus taurine vitamin E capsule is to mice half hemolysis value (HC50) impact
*: compare with 0g/kgBW group and have significant difference
From table 8, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, 0.75g/kgBW
Group compares with 0g/kgBW group, and half hemolysis value has significant difference (P < 0.05).I.e. Stichopus japonicus taurine vitamin E capsule exists
0.75g/kgBW group can improve the half hemolysis value of mice.
6 mice carbonic clearance experiments
By body weight from the india ink (0.05mL/10g) of mouse tail vein injection 4 times.Treat that prepared Chinese ink injects, immediately timing.Inject
After prepared Chinese ink 2,10min, take blood 20 μ L from angular vein clump respectively, and be added into 2mL0.1%Na2CO3In solution.With 755
Spectrophotometer is densitometric value (OD) at 600nm wavelength, with Na2CO3Solution makees blank.By sacrifice,
Take liver and spleen is weighed.Represent that the ability of mice carbonic clearance is calculated as follows a with carbonic clearance index (a):
K=(lgOD1-lgOD2)/(t2-t1)
A=body weight ÷ (liver weight+spleen weight) × k1/3
The carbonic clearance index of given the test agent group is significantly higher than the carbonic clearance index of matched group, can determine that this experimental result positive.
The impact on mice carbonic clearance ability of the table 9 Stichopus japonicus taurine vitamin E capsule
*: compare with 0g/kgBW group and have significant difference
From table 9, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, 0.25g/kgBW
Group compares with 0g/kgBW group, and carbonic clearance phagocytic index has significant difference (P < 0.05)；0.75g/kgBW group and 0g/kgBW
Group compares, and carbonic clearance phagocytic index has significant difference (P < 0.01).I.e. Stichopus japonicus taurine vitamin E capsule is at 0.25g/kgBW
The carbonic clearance ability of mice all can be improved with 0.75g/kgBW group.
7 Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell experiment (half intracorporal method)
Mouse peritoneal injection 20% (v/v, with normal saline) Sanguis Gallus domesticus cell (2000r/min, 10min) suspension 1mL,
Every 30min, cervical dislocation is put to death, is faced upward position and be fixed on Mus plate, through abdominal cavity saline injection 2mL, rotates Mus plate 1min.
Take peritoneal macrophage washing liquid mL, drip respectively on 2 microscope slides, put into the enamel box being lined with wet gauze, dislocation 37 DEG C
Incubator incubation 30min.Incubate complete, rinse in normal saline, to remove non-paster cell.Dry, with 1:1 acetone methanol solution
Fixing, the dyeing of 4% (v/v) Gicmsa-phosphate buffer is dried with distilled water rinsing again.Count under oil mirror, every counting 100
Individual macrophage, is calculated as follows phagocytic rate and phagocytic index:
Phagocyte × 100 of the macrophage number/counting of phagocytic percentage (%)=phagocytosis chicken red blood cell
The macrophage number of the chicken red blood cell sum/counting of phagocytic index=swallowed
The phagocytic percentage drawn carries out data conversion, X=sin-1 √ p the most as the following formula, and in formula, P is phagocytic percentage, decimally table
Show.The data obtained is measurement data, and the phagocytic percentage of given the test agent group and phagocytic index are all remarkably higher than the phagocytosis hundred of matched group
Divide rate and phagocytic index, can determine that this experimental result positive.
The impact on mouse macrophage phagocytosis chicken red blood cell phagocytic rate of the table 10 Stichopus japonicus taurine vitamin E capsule
*: compare with 0g/kgBW group and have significant difference
From table 10, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, 0.75g/kgBW
Group compares with 0g/kgBW group, and mouse macrophage phagocytosis chicken red blood cell phagocytic rate has significant difference (P < 0.01).I.e. Stichopus japonicus
Taurine vitamin E capsule can improve mouse macrophage phagocytosis chicken red blood cell phagocytic rate in 0.75g/kgBW group.
The impact on mouse macrophage phagocytosis chicken red blood cell phagocytic index of the table 11 Stichopus japonicus taurine vitamin E capsule
*: compare with 0g/kgBW group and have significant difference
From table 11, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, 0.75g/kgBW
Group compares with 0g/kgBW group, and mouse macrophage phagocytosis chicken red blood cell phagocytic index has significant difference (P < 0.01).I.e. sea
Ginseng taurine vitamin E capsule can improve mouse macrophage phagocytosis chicken red blood cell phagocytic index in 0.75g/kgBW group.
The mensuration (lactate dehydrogenase L DH algoscopy) of 8.NK cytoactive
Before experiment, target cell YAC-1 is carried out Secondary Culture by 24h, washes 3 times with Hank ' s liquid, with containing 10% calf before application
It is 4 × 105/mL that the RPMI1640 complete culture solution of serum adjusts cell concentration.Test mice cervical dislocation is put to death, aseptic
Take spleen, make splenocyte suspension, wash 2 times with Hank ' s liquid, be centrifuged 10min (1000r/min) every time.Abandoning supernatant will be thin
Endochylema is upspring, and adds 0.5mL aquesterilisa 20 seconds, adds 0.5mL2 times of Hank ' s liquid and 8mLHank ' s after splitting erythrocyte
Liquid, 1000r/min, 10min are centrifugal, resuspended with the 1mL RPMI1640 complete culture solution containing 10% calf serum, microscopy meter
Number, adjusting cell concentration with RPMI1640 complete culture solution is 2 × 107/mL.Make effect target than for 50:1.Take target cell and
The each 100 μ L of effector lymphocyte, add in U-shaped 96 well culture plate；Target cell Spontaneous release hole adds target cell and each 100 μ L of culture fluid,
Target cell maximum release aperture adds target cell and each 100 μ L of 1%NP40；Above-mentioned every it is all provided with three parallel holes, 37 DEG C, 5%CO2
Cultivating 4h in incubator, with 1500r/min, 96 orifice plates are centrifuged 5min, every hole is drawn 96 holes at the bottom of supernatant 100 μ L horizontalization and is cultivated
In plate, adding LDH matrix liquid 100 μ L, react 3-10min, then every hole adds the HCl solution 30 μ L termination of 1mol/L
Reaction, densitometric value (OD) at microplate reader 490nm, calculate NK cytoactive.
NK cytoactive (%)=(reacting hole OD-Spontaneous release hole OD)/(maximum release aperture OD-Spontaneous release hole OD) × 100
The NK cytoactive drawn carries out data conversion, X=sin-1 √ p as the following formula, and in formula, P is NK cytoactive, decimally
Represent.The data obtained is continuous data, and the NK cytoactive of given the test agent group is significantly higher than the NK cytoactive of matched group,
Can determine that this experimental result positive.
The impact on NK cells in mice activity of the table 12 Stichopus japonicus taurine vitamin E capsule
From table 12, per os gave the Stichopus japonicus taurine vitamin E capsule of mice various dose after 33 days, each dosage group NK
Cytoactive compares with 0g/kgBW group, and difference is all without there being significance (P > 0.05).I.e. Stichopus japonicus taurine vitamin E capsule pair
NK cells in mice activity is without impact.
To sum up, all experimental result judgment basis of embodiment 4 are:
" health food inspection and assessment technique specification " (2003 editions) regulation: at cellular immune function, humoral immune function, list
Core macrophage function, NK cytoactive any two aspect results of four aspects are positive, and can determine that this given the test agent has
Enhancing immunity function.Wherein two experimental results in cellular immune function assay project are the positive, or any one is real
Two the dosage group results tested are positive, can determine that cellular immune function assay result is positive.In humoral immune function mensuration project
Two experimental results are the positive, or two dosage group results of any one experiment are positive, can determine that humoral immune function measures knot
Fruit is positive.Two experimental results in mononuclear phagocyte functional examination project are the positive, or two agent of any one experiment
Amount group result is positive, can determine that mononuclear phagocyte function result is positive.More than one agent of NK cytoactive detection experiment
Amount group result is positive, can determine that NK cytoactive result is positive.
All the results shows of embodiment 4: per os gives the Stichopus japonicus taurine vitamin E capsule 33 of mice various dose
After it, comparing with 0g/kgBW group, this tested material can improve mice delayed allergy ability in 0.25g/kgBW group
(P < 0.05), improves the carbonic clearance ability (P < 0.05) of mice.Mice delayed allergy can be improved in 0.75g/kgBW group
Ability (P < 0.05)；Improve the lymphopoiesis ability (P < 0.01) of mice；Improve the antibody-producting cell number (P < 0.05) of mice,
Improve the half hemolysis value (P < 0.05) of mice, improve the carbonic clearance ability (P < 0.01) of mice, improve mouse macrophage and gulp down
Bite chicken red blood cell phagocytic rate (P < 0.01) and phagocytic index (P < 0.01).And Mouse Weight growth is had no adverse effects by tested material,
According to " health food inspection and assessment technique specification " (2003 editions) criterion to enhancing immunity health food,
Stichopus japonicus taurine vitamin E capsule enhancing immunity function results of animal is positive.
Claims (7)
1. the preparation method of a Stichopus japonicus functional health care product, it is characterised in that:
1) preparation of lyophilized Stichopus japonicus powder: be carried out by Stichopus japonicus, after roguing, by Stichopus japonicus through multistage enzymolysis, centrifugal, hyperfiltration treatment final vacuum lyophilization；
2) according to lyophilized Stichopus japonicus powder: taurine: the mass ratio of vitamin E is (180-220): (30-40): the ratio of (10-30) mixes；
3) by step 2) lyophilized Stichopus japonicus powder of gained, taurine powder, tocopherol admixture after 40-100 mesh sieves, put in mixer mixing 30min, obtain mixed powder.
Preparation method the most according to claim 1, it is characterized in that: step 1) described in by Stichopus japonicus through multistage enzymolysis method particularly includes: Stichopus japonicus is ground to form screened stock shape, according to Stichopus japonicus and water 1:(0.5-3) ratio mix, after crossing 100-200 eye mesh screen, holothurians autoenzyme, neutral protease, alkaline protease is utilized to carry out multi-stage degradation successively.
Preparation method the most according to claim 2, it is characterized in that: described holothurians autoenzyme, neutral protease, the use condition of alkaline protease be: holothurians autoenzyme, neutral protease, alkaline protease carry out multi-stage degradation at 40 DEG C, 45 DEG C, 55 DEG C respectively, described holothurians autoenzyme, neutral protease, the usage amount of alkaline protease are respectively, 0.1-2%, 0.1-0.6%, 0.02-0.07% in mass ratio.
Preparation method the most according to claim 1, it is characterised in that: step 1) described in centrifugal condition be: enzymolysis solution is centrifuged 10-40min by 3000-10000rpm.
Preparation method the most according to claim 1, it is characterised in that: step 1) described in Stichopus japonicus be the one in fresh and alive sea cucumbers, dry Stichopus japonicus, salted sea cucumber, and protein content is more than 50%.
6. utilizing the Stichopus japonicus functional health care product that the method described in claim 1 prepares, uniform color is consistent, and is grey-brown powder.
7. utilize the application in terms of improving body immunity of the health product described in claim 6.
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| Application Number | Title | Priority Date | Filing Date |
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| CN201610273191.8A Pending CN105942286A (en) | 2016-04-28 | 2016-04-28 | Sea cucumber functional health-care products and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110574769A (en) * | 2019-10-28 | 2019-12-17 | 烟台华康海洋食品有限公司 | sea cucumber nutritional product health product and preparation method thereof |
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| CN1593238A (en) * | 2002-08-30 | 2005-03-16 | 大连轻工业学院 | Sea cucumber mucopolysaccharide enriched food and its preparation method |
| CN101015361A (en) * | 2007-02-10 | 2007-08-15 | 中国海洋大学 | Instant sea cucumber infusion and its preparing process |
| CN101991148A (en) * | 2009-08-21 | 2011-03-30 | 东港市慧海海洋生物科技开发有限公司 | Sea cucumber capsules and preparation method thereof |
| CN102038226A (en) * | 2009-10-21 | 2011-05-04 | 东港市慧海海洋生物科技开发有限公司 | Hydrolysis instant sea cucumber and preparation method thereof |
| CN102754861A (en) * | 2012-07-31 | 2012-10-31 | 青岛海康水产发展有限公司 | Sea cucumber gelatin |
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- 2016-04-28 CN CN201610273191.8A patent/CN105942286A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1593238A (en) * | 2002-08-30 | 2005-03-16 | 大连轻工业学院 | Sea cucumber mucopolysaccharide enriched food and its preparation method |
| CN101015361A (en) * | 2007-02-10 | 2007-08-15 | 中国海洋大学 | Instant sea cucumber infusion and its preparing process |
| CN101991148A (en) * | 2009-08-21 | 2011-03-30 | 东港市慧海海洋生物科技开发有限公司 | Sea cucumber capsules and preparation method thereof |
| CN102038226A (en) * | 2009-10-21 | 2011-05-04 | 东港市慧海海洋生物科技开发有限公司 | Hydrolysis instant sea cucumber and preparation method thereof |
| CN102754861A (en) * | 2012-07-31 | 2012-10-31 | 青岛海康水产发展有限公司 | Sea cucumber gelatin |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110574769A (en) * | 2019-10-28 | 2019-12-17 | 烟台华康海洋食品有限公司 | sea cucumber nutritional product health product and preparation method thereof |
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